Identifying intrinsic and extrinsic determinants that regulate internal initiation of translation mediated by the FMR1 5' leader

<p>Abstract</p> <p>Background</p> <p>Regulating synthesis of the Fragile X gene (FMR1) product, FMRP alters neural plasticity potentially through its role in the microRNA pathway. Cap-dependent translation of the FMR1 mRNA, a process requiring ribosomal scanning through the 5' leader, is likely impeded by the extensive secondary structure generated by the high guanosine/cytosine nucleotide content including the CGG triplet nucleotide repeats in the 5' leader. An alternative mechanism to initiate translation – internal initiation often utilizes secondary structure to recruit the translational machinery. Consequently, studies were undertaken to confirm and extend a previous observation that the FMR1 5' leader contains an internal ribosomal entry site (IRES).</p> <p>Results</p> <p>Cellular transfection of a dicistronic DNA construct containing the FMR1 5' leader inserted into the intercistronic region yielded significant translation of the second cistron, but the FMR1 5' leader was also found to contain a cryptic promoter possibly confounding interpretation of these results. However, transfection of dicistronic and monocistronic RNA <it>ex vivo </it>or <it>in vitro </it>confirmed that the FMR1 5' leader contains an IRES. Moreover, inhibiting cap-dependent translation <it>ex vivo </it>did not affect the expression level of endogenous FMRP indicating a role for IRES-dependent translation of FMR1 mRNA. Analysis of the FMR1 5' leader revealed that the CGG repeats and the 5' end of the leader were vital for internal initiation. Functionally, exposure to potassium chloride or intracellular acidification and addition of polyinosinic:polycytidylic acid as mimics of neural activity and double stranded RNA, respectively, differentially affected FMR1 IRES activity.</p> <p>Conclusion</p> <p>Our results indicate that multiple stimuli influence IRES-dependent translation of the FMR1 mRNA and suggest a functional role for the CGG nucleotide repeats.</p

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition

Most eukaryotic mRNAs are monocistronic and translated by cap-dependent initiation. LINE-1 RNA is exceptional because it is naturally dicistronic, encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Here, we show that sequences upstream of ORF1 and ORF2 in mouse L1 function as internal ribosome entry sites (IRESes). Deletion analysis of the ORF1 IRES indicates that RNA structure is critical for its function. Conversely, the ORF2 IRES localizes to 53 nt near the 3′ end of ORF1, and appears to depend upon sequence rather than structure. The 40 nt intergenic region (IGR) is not essential for ORF2 IRES function or retrotransposition. Because of strong cis-preference for both proteins during L1 retrotransposition, correct stoichiometry of the two proteins can only be achieved post-transcriptionally. Although the precise stoichiometry is unknown, the retrotransposition intermediate likely contains hundreds of ORF1ps for every ORF2p, together with one L1 RNA. IRES-mediated translation initiation is a well-established mechanism of message-specific regulation, hence, unique mechanisms for the recognition and control of these two IRESes in the L1 RNA could explain differences in translational efficiency of ORF1 and ORF2. In addition, translational regulation may provide an additional layer of control on L1 retrotransposition efficiency, thereby protecting the integrity of the genome

The 5′ Leader of the mRNA Encoding the Mouse Neurotrophin Receptor TrkB Contains Two Internal Ribosomal Entry Sites that Are Differentially Regulated

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5′ leaders (1428 nt and 448 nt), both of which include the common 3′ exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5′ leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5′ leader are differentially regulated, in part by PTB1

The mouse TrkB 5′ leaders are able to initiate translation when cap-dependent translation is inhibited.

<p>A) A dual monocistronic construct containing the pGL3 multiple cloning site upstream of the <i>Renilla</i> luciferase and the pGL3 multiple cloning site, EMCV, or the TrkB 5′ leaders upstream of the <i>Photinus</i> luciferase gene was co-transfected into C6 cells with a construct encoding for a hypophosphorylated 4EBP construct or a null vector. The level of <i>Photinus</i> luciferase activity from each mRNA, when co-transfected with the null vector, was normalized to 100 percent. The <i>Renilla</i> and <i>Photinus</i> luciferase activity obtained in cells co-transfected with hypophosphorylated 4EBP is represented as a percentage of the activity obtained in cells co-transfected with the null plasmid. B) Monocistronic <i>Photinus</i> luciferase mRNA containing the β-globin or TrkB 5′ leaders was <i>in vitro</i> translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. <i>Photinus</i> luciferase activity for each mRNA in the absence of cap analog was set to 100 percent.</p

The mouse TrkB 5′ leaders exhibit IRES activity when expressed in dicistronic RNA constructs.

<p>Dicistronic luciferase mRNA containing the β-globin and the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio for each construct was normalized to that of the negative control, β-globin.</p

The mouse TrkB 5′ leaders exhibit an increased <i>Photinus</i> to <i>Renilla</i> luciferase (P∶R) ratio.

<p>Dicistronic luciferase constructs containing the β-globin, EMCV, and TrkB 5′ leaders were transfected into the C6 and N2a neural cell lines. The P∶R ratio from each construct was normalized to that obtained from the β-globin construct, whose P∶R ratio was set to one.</p

PTB increases IRES activity from the Ex2 IRES, but does not affect Ex1a IRES activity.

<p>A) Dicistronic luciferase mRNA containing the Ex1a, Ex2, or β-globin 5′ leader was <i>in vitro</i> translated in RRL that was either untreated or supplemented with 0.4 mg of PTB1. The change in the P∶R ratio for the samples when in the presence of PTB1 relative to the untreated sample are shown. B) The two mouse TrkB IRESes demonstrate differential regulation. SH-SY5Y cells treated with 2 mM retinoic acid or with DMSO (mock) for four days were transfected with dicistronic mRNA containing the β-globin, Ex2, or Ex1a 5′ leaders. The P∶R ratios were normalized to that from the mRNA containing the β-globin 5′ leader.</p